The present invention relates to a method for producing prophylactically or therapeutically active proteins and to the means employed therefor, in particular syringes.
Therapeutically active proteins such as erythropoietin, insulin or interferons have been known for a long time. Many of these proteins have already been approved as medicaments and are accordingly employed frequently. Because of the large costs associated with the development and approval of these medicaments, however, there is a need for simple and cost-effective alternatives for the preparation of therapeutically active proteins. In addition, not all therapeutically active proteins are approved as medicaments. Nevertheless, however, there is frequently a need for these proteins also to be administered to the patient. Particularly important in this connection, because they are presumed to be well tolerated by the body, are autologous proteins, that is to say those intrinsic to the body. These proteins include the interleukin 1 receptor antagonist, interleukin 4, interleukin 10 and the type I or type II tumor necrosis factor receptor. Proteins intrinsic to the body moreover have the advantage that the natural post-translational modifications such as glycosylations are already present. This is not the case with most normally obtainable recombinant proteins because they are produced in prokaryotic hosts.
Stimulation of monoctyes by adherent immunoglobulin G to form the interleukin 1 receptor antagonist is described by Arend and Leung in Immunological Reviews (1994) 139, 71-78 and Moore et al. in Am. J. Respir. Cell Mol. Biol. (1992) 6, 569-575. Andersen et al. in Autommunity (1995) 22, 127-133 explains that the therapeutic effect of immunoglobulin G to be observed in vivo cannot be attributed to an enhanced formation of interleukin 1 receptor antagonist, and that the in vitro formation of the interleukin 1 receptor antagonist (IRAP, IL-1Ra) by monocytes depends on serum and plasma constituents adsorbed on polypropylene. The therapeutic use of adsorbed serum and plasma constituents to stimulate the formation of therapeutically interesting proteins in therapies is not only very costly but also involves the risk of contamination with infectious particles with which the serum and plasma constituents may be contaminated. Methods for producing IL-1Ra which can be employed directly in the therapy without using adsorbed serum and plasma constituents are not described in the aforementioned publications.
The technical problem on which the present invention is based is thus to provide methods and means for producing IL-1Ra which serve as safe, cost-effective alternatives which can be carried out quickly for the use and for the production of conventional pharmaceutical products.
The invention solves this problem by providing a method for producing IL-1Ra in a syringe made of glass, quartz or a plastic, the syringe being filled with a body fluid from an organism, for example a human or animal body, and incubated, and the IL-1Ra being formed.
A preferred embodiment of the present invention provides for the internal structure of the syringe to consist of a special material, in particular a glass, plastic, quartz and/or corundum, whose surface is, in a particularly preferred embodiment of the invention, modified, in particular with the aid of a corrosive agent, for example an acid or an alkali, in particular chromosulphonic acid, and subsequently, after removal of the agent and washing the syringe where appropriate, that is to say in a particularly preferred manner, the surface of the internal structure of the syringe is sterilized, in particular by autoclaving. The syringe is then filled with a patient""s body fluid and incubated, and IL-1Ra is formed thereby. The body fluid enriched with the protein can then be reinjected into the patient, for example into a diseased joint. However, to increase the purity of the IL-1Ra which has been formed, the body fluid is preferably centrifuged and the supernatant is sterilized by filtration, divided into aliquots and stored for a later treatment. The invention thus provides in a preferred embodiment in a first step of the method for the surface of the internal structure of the syringe to be modified, in particular with the aid of a corrosive agent, such as an acid or an alkali, in particular chromosulphonic acid, and then, if desired, the surface of the internal structures of the syringe is sterilized, in particular by autoclaving. A drying can be provided before and/or after the sterilization. After the modification and the sterilization which takes place where appropriate, the syringe is filled in a second step of the method with a body fluid, in particular blood, lymph, saliva or urine, and incubated. The body fluid is preferably taken from the patient directly with the syringe. The, preferably modified, surface of the internal structure of the syringe induces in the body fluid specifically, to an extent which differs quantitatively depending on the material employed, the internal structure of the syringe, modification employed, in particular etching of the internal structure, sterilization employed, in particular autoclaving, and body fluid employed, the formation of IL-1Ra which, accordingly, becomes enriched or is formed in the body fluid present in the syringe. The body fluid enriched in this way can be stored sterile in the syringe and be returned to the patient as required directly without further treatment or, preferably, after centrifugation and/or sterilization by filtration.
The invention thus also relates to a method for the prophylactic or therapeutic treatment of the human or animal body, for example for the treatment of rheumatism, osteoarthritis and/or back symptoms, wherein a body fluid, for example blood, is taken from the human or animal body by means of a syringe according to the present invention, this body fluid is incubated in the syringe, and IL-1Ra is formed or enriched thereby, and the body fluid is returned using this syringe to the same or a different human or animal body.
The IL-1Ra which is formed can advantageously be modified, for example glycosylated. It is self-evident that the invention also encompasses the formation of other modifications or variants of IL-1Ra, such as truncated forms, mutants or other derivatives.
In conjunction with the present invention, an internal structure of a syringe means any region or any structure of the syringe which is present in its interior, that is to say in the sample reception region and may come into contact with the body fluid to be received. The internal structure of a syringe is particularly advantageously its inner surface, preferably a surface with a texturing to increase the surface area. It is self-evident that the present invention can also be carried out using a commercially available syringe without a special configuration in its internal cavity. In such a case the internal structure is the inner surface of the syringe barrel and the part of the piston present in the barrel. The internal structure can, in a particularly preferred embodiment, additionally be formed by articles introduced into the interior of the syringe, such as particles, spheres, beads, gels, glass wool, corundum, quartz, sand, plastic or glass granules or powder or the like in order to enlarge the internal surface area of the syringe and thus provide a larger inducing surface. The material of which the internal structure consists or which is present in the internal structure can be a material different from that of which the remainder of the syringe consists. For example, the syringe may consist of plastic and parts of its internal structure may, for example, consist of glass granules.
Additional structures of these types, such as, for example, glass beads with a diameter of from 1 to 5 mm, should, according to a preferred embodiment of the invention, however occupy not more than 50% of the internal volume of the syringes employed. The syringes employed may be, for example, 10 to 100 ml syringes.
In connection with the present invention, a modified surface of an internal structure of the syringe means a surface which has been treated with a corrosive agent and which is capable of inducing and/or enhancing the formation of IL-1Ra in a body fluid of a human or animal. The modified surface may be distinguished by a particularly great cleanliness, that is to say substantial or complete absence of contaminants and/or a chemical/physical modification of the surface properties and/or structure. The modified surface of the internal structure is preferably produced by employing an alkali or an acid, for example chromosulphonic acid, in particular 20 to 80% strength, particularly preferably 50% strength, chromosulphonic acid. The syringe is preferably incubated with the corrosive agent, in particular the chromosulphonic acid, for 5 to 30 min.
After removal of the agent it is possible and preferred to carry out one or more washing steps and, where appropriate, preferably a sterilization, for example by autoclaving, in particular autoclaving at 100xc2x0 C. to 150xc2x0 C. for 20 to 60 min under a pressure of from 1 to 5 bar. After the washing and before and/or after the autoclaving it is possible where appropriate also for one or more drying steps to take place at 60 to 150xc2x0 C., preferably 80xc2x0 C., for 30 to 120 min in, for example, a drying oven.
A particularly advantageous configuration of the invention provides for the syringe, in particular the internal structure of the syringe, to be produced from glass, for example quartz glass, corundum, quartz, a plastic such as polystyrene, polyethylene, polyvinyl chloride, polypropylene, or a similar material, that is to say consists of these materials or essentially comprises these materials or mixtures thereof.
It has surprisingly been possible to show within the framework of the present teaching that a syringe made of glass, and/or an internal structure made of glass, in particular glass granules, displays a particularly strongly inducing effect.
In a particularly preferred embodiment, heated glass is used before the treatment, that is to say before removal of the body fluid, in particular glass which has been heated to 100xc2x0 C.-210xc2x0 C., in particular 170xc2x0-200xc2x0 C., preferably 180xc2x0 C., and which is, of course, cooled before the use according to the invention. In a particularly preferred embodiment, the glass can be in the form of glass powder or glass granules.
The internal structures may, however, also consist of quartz, for example quartz powder or quartz sand and/or corundum, for example in the form of a suspension in water, or comprise the latter.
In a particularly preferred embodiment, these materials display IL-1Ra-inducing properties after modification, in particular etching, has taken place where appropriate and after the sterilization has taken place where appropriate.
A further preferred embodiment of the invention provides for the internal structure of a syringe to be provided with a coating which can be modified and can be sterilized, and for the modification and, where appropriate, the sterilization to be carried out subsequently.
The present invention is advantageous inasmuch as it provides a method which is simple to carry out and with which it is possible to produce autologous IL-1Ra which can be produced by induction, in particular surface induction, and can, in the form thus produced, that is to say together with the other constituents of the fluid present in the syringe, be readministered to the patient directly, that is to say without further manipulation such as, for example, transfer into another container. In a preferred embodiment, a centrifugation and/or sterilization by filtration can be provided to remove solid constituents before the IL-1Ra which has been formed is administered. The use of commercially available, often costly, medicaments thus becomes redundant. Finally, the invention proves to be advantageous since it avoids contamination, pollution, infection or the like of the IL-1Ra which derives from a medicament production taking place outside the patient.
The present invention thus provides in a particularly preferred embodiment a method for producing the interleukin 1 receptor antagonist, where the internal structures of the syringe consist of a specially treated material, in particular glass, quartz or plastic, where the surface of the internal structures of the syringe has been modified, in particular with the aid of a corrosive agent, in particular chromosulphonic acid, where the surface of the internal structures of the syringe has, where appropriate, subsequently been sterilized, in particular by autoclaving, and where the syringe is filled with a body fluid, preferably blood, and incubated, and IL-1Ra is formed and enriched in the body fluid. Inter alia because of the special modification of the surface of the internal structures of the syringe, in particular etching, and the special sterilization of the surface, in particular autoclaving, the syringe of the invention is able to stimulate the monocytes present in the blood to form IL-1Ra so that the latter becomes enriched in the blood. The blood present in the syringe can, in one embodiment of the present invention, after incubation, that is to say after enrichment of the IL-1Ra, be returned to the patient from whom the blood introduced into the syringe was taken, directly, without further manipulation such as, for example, transfer.
It is possible and advantageous in a particularly preferred embodiment of the present invention to provide a centrifugation and/or sterilization by filtration of the blood present in the syringe in order to remove solid constituents such as cells. It is then divided into aliquots where appropriate. It is thus also provided by the invention that the blood can be taken from the patient by means of the syringe equipped with a specially modified and sterilized, in particular autoclaved, surface of the internal structures, the blood can be incubated in the syringe and, after formation of IL-1Ra, can be returned to the same patient or another one with the syringe. Such a procedure is particularly advantageous for example in the area of neuroorthopedics, that is to say, for example, for back symptoms with a neurological causation. Suitable treatment for such symptoms to date has been only an intervertebral disk operation, cortisone treatment, irrigations with saline solutions or the like. The provision by the invention of IL-1Ra, that is to say, in particular, autologous IL-1Ra, also makes treatment of rheumatism and osteoarthritis possible in a particularly simple, cost-effective and efficacious way.
The invention provides in another embodiment for the internal structure of the syringe additionally to be coated with anticoagulants, in particular heparin, citrate, EDTA, CPDM or CPDA. It has surprisingly been possible to show within the framework of the present teaching that a good induction is achieved on use of heparin as anticoagulant. It is also possible to provide according to the invention for the anticoagulants to be employed not as coating but unbound in the container, for example put in the lyophilized or liquid state into the syringe.
However, in a particularly preferred embodiment of the present invention, no anticoagulant, in particular no heparin, is employed in the syringe. This leads to a further improvement in the incubation.
The invention provides in a further preferred embodiment for the incubation of the body fluid in the syringe to be carried out over a period of from 12 to 72 hours, preferably 24 hours, preferably at room temperature, that is to say 20xc2x0 C. to 41xc2x0 C., in particular at 37xc2x0 C.
The invention also provides in one configuration of the invention for the body fluid to be treated further after formation of the therapeutically or prophylactically active protein in the body fluid, in order, for example, to remove particular constituents of the latter, for example blood plasma or blood platelets. This removal can in a preferred embodiment of the invention be carried out by centrifugation or filtration.
The invention relates in a further embodiment to a method for producing a syringe which is suitable for the in vitro induction of prophylactically or therapeutically active proteins, in particular the interleukin 1 receptor antagonist, where the syringe is distinguished by the specially treated material of the internal structure of the syringe, in particular plastic or glass. The invention provides in particular for the surface of the internal structure of the syringe to be etched by a corrosive agent, in particular an alkali or an acid, in particular using chromosulphonic acid. After removal of the corrosive agent and washing of the syringe it can be provided for the surface of the internal structure to be sterilized, in particular autoclaved. It can preferably also be provided for a syringe, preferably produced from glass, to be heated before use thereof, for example to 100xc2x0 C. to 210xc2x0 C., in particular 170xc2x0-200xc2x0 C.
The invention of course also relates to the syringe produced in this way, which, in a particularly preferred embodiment, is produced from glass, quartz or plastic, in particular polystyrene, polyvinyl chloride, polyethylene or polypropylene, where the syringe is distinguished by the special treatment of the surface of the internal structure of the syringe, in particular produced from glass, quartz or plastic, which is carried out by exposure to a corrosive agent. In a preferred manner, the syringe has been heated before use thereof, preferably to 100xc2x0 C.-210xc2x0 C., for example 170xc2x0 C.-200xc2x0 C.
The invention also relates to the use of alkali or acid, in particular chromosulphonic acid, for modifying the surface of the internal structures of the syringes of the invention, preferably made of polystyrene,polyvinyl chloride, polyethylene, polypropylene, quartz or glass, for the in vitro induction of prophylactically or therapeutically active proteins, preferably the interleukin 1 receptor antagonist.
The invention also relates to the use of devices or substances which enlarge the surface area, such as glass powder, glass granules, quartz powder, quartz sand, corundum, spheres, beads, sand and so forth for use in an aforementioned method, that is to say in particular for use as inducer of IL-1Ra formation in a vessel, for example a syringe.
Further advantageous configurations of the invention are evident from the dependent claims.
The invention is illustrated in detail by means of figures and exemplary embodiments.